Primer-dependent nucleic acid amplification reactions, which may include detection of amplification products (“amplicons”), require “specificity,” that is, annealing of a primer to the intended place in a nucleic acid strand and extension of primers bound only to the intended target sequence. Conventionally, specificity is obtained by making a primer sufficiently long so that under the amplification reaction conditions, primarily during the primer-annealing step, the primer goes to only one place in a nucleic acid strand.
Certain amplification reactions are intended to distinguish between or among allelic variants, for example, single-nucleotide polymorphisms (SNPs). One way to do that is to amplify all variants and to distinguish between or among them by allele-specific hybridization probes such as molecular beacon probes. For such an approach, the amplification primers are made equally complementary to all variants so as to amplify a region that includes the sequence that varies between or among alleles, and a probe identifies an allele that is present in the amplified product or products. See, for example, Tyagi et al. (1998) Nature Biotechnology 16:49-53. If the sequence being investigated is an allele, such as a SNP that is present in a mixture with another allele, for example, a wild-type (WT) variant, distinguishing by use of a probe has a practical detection limit of about 3% (not less than about 30,000 target allele molecules in the presence of 1,000,000 molecules of the alternate allele) due to the tendency of amplification of the prevalent allele to overwhelm amplification of the rare allele.
Another way to distinguish between or among alleles is to use a primer that is selective for the sequence being investigated. For such an approach, the primer is made complementary to the sequence that varies between or among alleles, and amplified product may be detected either by labeled primers, a DNA binding dye, or a labeled probe (in this case the probe detects a sequence common to amplicons of all alleles). A primer that is highly specific typically has a length of 15-30 nucleotides. Such a conventional primer has very limited selectivity for one allele over another. It is known that shortening a primer will improve its selectivity, but because that improvement comes at the expense of specificity, and because short primers are unlikely to form stable hybrids with their target sequence at typical annealing temperatures, shortening a primer is of limited value for analyzing mixtures of alleles.
Other modifications of primers have been developed to improve their selectivity while retaining specificity. One such approach is ARMS (“amplification refractory mutation system”). An ARMS primer has a 3′-terminal nucleotide that is complementary to the sequence variant being investigated, but that is mismatched to another allele or alleles. See Newton et al. (1989) Nucleic Acids Res. 17:2503-2516; and Ferrie et al. (1992) Am. J. Hum. Genet. 51:251-262. ARMS relies on the refractory nature of certain DNA polymerases, that is, a tendency not to extend a primer-target hybrid having such a mismatch. ARMS has been demonstrated to be useful for determining zygosity (homozygous WT, heterozygous, or homozygous mutant (MUT)), but it has a practical detection limit for other uses of about 1% (not less than about 10,000 target allele molecules in the presence of 1,000,000 molecules of the alternate allele).
Another approach is to make a primer into a hairpin to increase its selectivity. See Tyagi et al. European patent EP 1 185 546 (2008), which discloses making the hairpin loop complementary to the sequence being investigated but mismatched to another allele or alleles; and Hazbón and Alland (2004) J. Clin. Microbiol. 42:1236-1242, which discloses making the terminal nucleotide of the 3′ arm of the hairpin primer complementary to the sequence variant being investigated but that is mismatched to another allele or alleles, as with ARMS. These modifications also have practical detection limits of about 1% (not less than about 10,000 target allele molecules in the presence of 1,000,000 molecules of the alternate allele).
Jong-Yoon Chun and his colleagues at the Seegene Institute of Life Science in Seoul, South Korea, have devised a type of primer that they refer to as a “dual-priming oligonucleotide (DPO).” See, Chun et al. (2007) Nucleic Acids Res. 35 (6) e40; Kim et al. (2008) J. Virol. Meth. 149:76-84; Horii et al. (2009) Lett. Appl. Microbiol. 49:46-52; WO 2006/095981 A1; and WO 2007/097582 A1. A DPO primer consists of three segments: a long 5′ high-temperature segment, for example, 20-25 nucleotides in length, a central separation segment of five deoxyriboinosines, and a 3′ priming segment, generally 8-12 nucleotides in length, that is complementary to the intended target sequence but mismatched to other target sequences. The target sequence is complementary to all three segments, but the Tm of the 3′ segment is lower than the Tm of the 5′ segment, due to its shorter length, and the separation segment has the lowest Tm due to the five deoxyriboinosines. A DPO primer is designed such that amplification results only if both the 5′ segment and the 3′ segment hybridize to a target strand. According to Chun et al. (2007), the separation segment was selected to be five deoxyriboinosines, because 3-4 and 6-8 deoxyriboinosines did not give results as good; the 3′ segment was positioned so as to provide a GC content of 40-80%, and the 5′ segment was provided a length sufficient to raise its Tm above the annealing temperature to be used in 3′-RACE amplifications (Nucleic Acids Res. 35(6) e40 at page 2). Chun et al. reports successful genotyping (homozygous wild type, heterozygous, or homozygous mutant) of a SNP (G→A mutation) in the CYP2C19 gene using two pairs of DPO primers. Of the four DPO primers, one had a 3′ segment 12-nucleotides long, perfectly complementary to both alleles; one had a 3′ segment 9-nucleotides long, perfectly complementary to both alleles; and two had 3′ segments 8-nucleotides long with the variable nucleotide located in the middle, that is, at the fourth nucleotide position from the 3′ end. Genotyping was accomplished by means of gel electrophoresis.
There are situations in which it is desired to detect a very rare first allele in the presence of a very abundant second allele. This has been termed “sensitivity”. In other words, the primer must not only be “specific” (go to the correct place in the genome), and be “selective” (reject wild type or other abundant sequences similar to the target sequence), but it must be highly selective, that is, “sensitive” enough to detect a very few mutant or other rare first sequence in the presence of an abundance of wild type or other abundant second sequence. See Makarov and Chupreta international patent application WO 2012/112 582 A2 at paragraph [0004].
To improve sensitivity while retaining specificity and selectivity, Vladimir Makarov and his colleagues at Swift Biosciences (Ann Arbor, Mich., U.S.A.) disclose a “discontinuous polynucleotide [“primer”] design” (WO 2012/112 582 A2 at paragraph [0051]) that has been commercialized as myT™ Primers. Such primers may be viewed as long conventional primers that are composed of two oligonucleotides so as to create an eight-nucleotide 3′ priming sequence; and adding complementary tails to the 5′ end of that sequence and to the 3′ end of the other oligonucleotide to form a high-temperature stem. Through the stem, the two oligonucleotides are joined non-covalently and form a stable three-way junction when bound to the target sequence. The oligonucleotide with the eight-nucleotide 3′ end is referred to as the “primer”, and the other oligonucleotide is referred to as the “fixer”. The function of the fixer is to provide specificity, that is, to bind the primer to the intended place in the genome. It is accordingly long, typically about 30-nucleotides in length. The function of the tails is to hybridize the two oligonucleotides under amplification conditions, so the tails also are fairly long, forming a stem 20-25 nucleotides in length. The function of the eight-nucleotide 3′ region is to prime with selectivity. The discontinuous hybridization “in effect stabilized binding between the [priming] region of the primer oligonucleotide even if this region is as small as eight bases, thereby increasing the efficiency of PCR.” (WO 2012/112582 A2). Further improvements are disclosed in Examples 9-11 of WO 2012/112582 A2. The nucleotide that is mismatched to the wild-type target is made the 3′-terminal nucleotide, as in ARMS; a third oligonucleotide, a blocking oligonucleotide (“blocker”), whose 5′-terminal nucleotide overlaps the 3′-terminal nucleotide of the primer and is complementary to the wild-type target, is included in the amplification reaction; and the 3′-terminal nucleotide of the primer is made of locked nucleic acid (“LNA”). For the detection of single-nucleotide polymorphisms in the K-ras and B-raf genes, detection sensitivity of one mutant in 14,000 wild-type (approximately 0.01%) was disclosed.
There remains a need for a single-oligonucleotide primer that has the ability to detect and, preferably, to quantify the number of a rare first target sequence, for example, a mutant target sequence, in the presence of a very large number of a second target sequence that differs from the first target sequence by as little as a single nucleotide, for example, a wild-type sequence.